Thermophilic ethanol production in a two-stage closed system

ABSTRACT

Production of ethanol by a process in which a strain of Bacillus stearothermophilus or other thermophilic, facultatively anaerrobic bacterium is selected for the characteristics of fermenting sugars both aerobically and anaerobically and of being active in anaerobic fermentation at 70° C. or above and (i) anaerobic fermentation is carried out with continuing removal of ethanol at 70° C. or above; (ii) the fermentative activity of the bacterium is maintained by withdrawing a proportion of the anaerobic fermentation medium on a continuing basis preferably with removal of ethanol and allowing the bacteria therein to multiply aerobically, using residual sugars or metabolites thereof present in the medium, before being returned to the anaerobic fermentation.

This application is a continuation of application Ser. No. 435,480,filed Nov. 27, 1989, now abandoned.

FIELD OF INVENTION

The invention relates to alcohol, that is to say ethanol, production byfermentation.

GENERAL DISCUSSION

Alcohol production from waste or by-product sugars whether arising assuch or derived from conversion of other carbohydrates has long beenknown but is currently of growing importance. Cheap oil at present, andsevere food shortages in particular regions, cannot detract from thebasic unsoundness of relying on non-renewable energy sources when,properly managed, agriculture could provide food and energy world wide.

We have studied known alcohol production processes, largely by yeasts,and concluded that a key improvement in economic operation, ifachievable in practice, is use of temperatures at which the alcohol canconveniently be removed directly as vapour from the fermentation medium.Yeasts of course are incapable of growth at such temperatures, and wehave turned to thermophilic bacteria.

Yeasts ferment only glucose, maltose or sucrose whereas some bacteriacan also utilise cellobiose from enzymic hydrolysis of cellulose orxylose and arabinose from hydrolysis of hemicellulose. The latter(pentose) sugars are the major components of waste streams frompaper-making or from pretreatments of straw such as steam-explosion ordilute acid hydrolysis. The economics of ethanol production from sugarcane would for example be greatly improved if the bagasse could be soutilised as well as the juice.

Some thermophiles have been described which can utilise all these sugarsto produce high yields of ethanol e.g. Clostridiumthermosaccharolyticum, Cl. thermohydrosulfuricum or Thermoanaerobacterethanolicus. However they are strict anaerobes and their reportedproperties compare unfavourably with the Bacillus stearothermophilusstrains described below. Moreover we have seen that facultativeanaerobes have additional advantages by allowing a novel mixedaerobic-anaerobic process which allows by-products of the anaerobicphase to be utilised aerobically to regenerate catalytic biomass.

Most facultative anaerobes do not produce high yields of ethanol. Inprevious disclosures we described a `metabolic steering` strategywhereby mutants believed to be of Bacillus stearothermophilus NCA 1503may be manipulated to make high yields of ethanol ¹, 2. That strategyinvolved eliminating L-lactate production by selecting mutations inL-lactate dehydrogenase. The resulting mutant was expected to makeacetate, ethanol and formate anaerobically in yields of 2:2:4 per moleof sucrose. Surprisingly however yields of ethanol were higher than thistheoretical maximum under certain conditions, notably low pH and highertemperatures, and this was ascribed to a catalytic conversion of sucroseto ethanol+CO₂ during a final non-growth stage in batch cultures.

We have now discovered that the results previously reported are to anextent in error in that the organism described is not a derivative of B.stearothermophilus NCA 1503 (NCIB 8924) as we assumed (Payton andHartley 3) nor indeed of any specific known thermophilic bacillus.Instead it appears to be derived from a novel strain of the speciesBacillus stearothermophilus that has properties that make it muchsuperior to known strains for the purposes described above. Inparticular, it has a much higher growth rate than strain NCA 1503 bothaerobically and anaerobically at temperatures above 60° C. and growsanaerobically above 70° C., at which temperature growth ceases withstrain NCA 1503. Moreover, it utilises both cellobiose and the pentosesugars found in a crude dilute acid hydrolysis of wheat straw producedby the ICI process described by Ragg and Fields ⁴.

Hence though this invention is not restricted to particular bacteria, itconcerns facultative anaerobes such as B. stearothermophilus strainLLD-R (NCIB deposit details below) that rapidly ferment a wide range ofsugars including cellobiose and pentoses both aerobically andanaerobically above 70° C. Such strains would normally produce lactateanaerobically, but this pathway is eliminated by selecting mutations inNAD-linked lactate dehydrogenase. Moreover acetate production may besuppressed by physiological controls such as acid pH, highertemperatures or high levels of extracellular acetate, or by furthergenetic lesions in enzymes of the acetate pathway. This leads to achannelling of anaerobic metabolism via pyruvate dehydrogenase,resulting in conversion of sugars to ethanol+CO₂. The resulting cellsmay not grow anaerobically but can catalyse conversion of sugars toethanol without growth.

Hence an aspect of this invention is a process in which such cells areused in a catalytic anaerobic production stage fed with sugars butminimizing growth. Most of the ethanol is automatically removed into thevapour phase above 70° C., so the production phase can be fed with highsugar concentrations without exceeding the ethanol tolerance of theorganism (ca. 4% w/v). We have seen that these properties lendthemselves to a novel continuous process in which the optimum fermenterproductivity is achieved by continuous cell recycle after removingaqueous phase products by centrifugation or filtration. The minimumgrowth rate necessary to maintain catalytic viability can be achieved bybleeding off a small proportion of cells during recycle; this results inconversion of an equivalent proportion of the influent sugar into freshbiomass. Alternatively, the remaining aqueous phase ethanol is removedbefore returning spent cells, unhydrolysed sugars, residual traces ofethanol and by-products such as acetate and formate to an aerobic`biomass` stage. The aerobic cells are then returned to the catalytic`production` stage, if necessary through an intervening anaerobic`adaptation` stage. An attractive feature of such a reactor conformationis that automatic process control to optimise ethanol yield can bemaintained by minimizing aerobic CO₂ and maximizing anaerobic CO₂.

HISTORY AND CHARACTERISTICS OF STRAINS

Bacillus stearothermophilus strain LLD-15 (NCIB deposit details below)arose during attempts to obtain mutants of Bacillus stearothermophilusstrain NCA 1503 lacking L-lactate dehydrogenase activity by selectingfor suicide substrate resistance (Payton and Hartley ³). It wasnaturally assumed to be a mutant of the latter strain, but in fact it isderived from a novel extreme thermophile, Bacillus stearothermophilusstrain LLD-R. Strain LLD-R arises spontaneously and reproducibly fromreversion of strain LLD-15 and is selected on plates or duringcontinuous cultures under which it grows more rapidly, e.g. at low pH inmedia containing sugars+acetate and formate. It produces L-lactateanaerobically and contains high levels of L-lactate dehydrogenase so itis clearly a `wild-type` revertant of the LLD-15 lesion.

Both the mutant B. stearothermophilus strain LLD15 and the wild-type,strain LLD-R, are gram positive, spore-forming rods that resemble thebroad class Bacillus stearothermophilus in morphology and growthtemperature range. However in a series of biochemical and growth tests(Table 1) they differ from B. stearothermophilus NCA 1503 and all otherrelated strains in the extensive collection of Sharp et. al. J. Gen.Microbial 117 201 (1980). These properties merit a generalclassification as Bacillus stearothermophilus following Donk, L. (1920)J. Bacteriol. 5 373, but the growth temperature range is distinctlyhigher than that for strain NCA 1503, from which the mutant was thoughtto derive. Hence both organisms are deposited as novel type strainsBacillus stearothermophilus LLD-R (NCIB 12403) and Bacillusstearothermophilus LLD-15 (NCIB 12428) with the National Collection ofIndustrial and Marine Bacteria, Torry Research Station, P.O. Box 31,Aberdeen, AB9 8DG, Scotland. The respective dates of deposit are Feb.10, 1987 and Apr. 9, 1987.

Strains LLD-R and LLD-15 grow well on a rich BST medium (g/1): tryptone(Oxoid) 20.0; yeast extract (Oxoid) 10.0; K₂ SO₄, 1.3; MgSO₄.7H₂ O,0.27; MnCl₂ 4H₂ O, 0.015; FeCl₃.6H₂ O, 0.007; citric acid, 0.32;supplemented with an appropriate carbon source and adjusted to therequired pH with KOH or H₂ SO₄. However we have also developed a fullydefined Medium BST-MM (g/1): C source variable; K₂ SO₄, 0.3; Na₂ HPO₄,1.0; MgSO₄, 0.4; MnCl₂.4H₂ O, 0.003; CaCl₂, 0.005; NH₄ Cl, 1.0; citricacid, 0.16; methionine, 0.2; (mg/1): nicotinic acid, 10; biotin, 10;thiamine, 10; ZnSO₄.7H₂ O, 0.4; boric acid, 0.01; CoCl₂.6H₂ O, 0.05;CuSO₄.5H₂ O, 0.2; NiCl.sub. 3.6H₂ O, 0.01; EDTA, 0.25.

                  TABLE 1                                                         ______________________________________                                        Comparison of new strains with other thermophilic bacilli                     ______________________________________                                                                       B. stearo.                                                                           B. stearo.                                           B. stearo.                                                                             B. stearo.                                                                             NCA    ATCC                                                 LLD-15   LLD-R    1503   12016                                   ______________________________________                                        Starch hydrolysis                                                                          R        ..       +      +                                       Casein hydrolysis                                                                          +        ..       +      w                                       Gelatin hydrolysis                                                                         +        ..       +      w                                       Hippurate hydrolysis                                                                       +        ..       -      ..                                      Citrate utilisation                                                                        -        -        -      ..                                      Catalase     +        +        -      -                                       Oxidase      -        -        -      +                                       Growth in 3% saline                                                                        -        ..       w      -                                       Sugar fermentations:                                                          Galactose    -        -        w      +                                       Glycogen     -        ..       +      w                                       Mannitol     -        -        -      w                                       Raffinose    -        -        +      -                                       Starch       -        ..       +      -                                       Trehalose    +        ..       +      -                                       Xylose       +        +        -      +                                       ______________________________________                                                     B. stearo.                                                                             B. stearo.                                                                             B. stearo.                                                                           B. stearo.                                           240      262      RS93   126                                     ______________________________________                                        Starch hydrolysis                                                                          R        R        -      -                                       Casein hydrolysis                                                                          +        +        -      -                                       Gelatin hydrolysis                                                                         -        +        -      w                                       Hippurate hydrolysis                                                                       ..       ..       ..     ..                                      Citrate utilisation                                                                        ..       ..       ..     ..                                      Catalase     +        +        +      +                                       Oxidase      +        +        +      +                                       Growth in 3% saline                                                                        w        +        +      w                                       Sugar fermentations:                                                          Galactose    -        -        -      -                                       Glycogen     +        +        -      -                                       Mannitol     -        -        +      +                                       Raffinose    -        -        -      -                                       Starch       +        w        -      -                                       Trehalose    +        -        w      w                                       Xylose       -        -        -      -                                       ______________________________________                                                                           B.                                                      B. stearo. 10                                                                            B. caldotenax                                                                            caldovelox                                 ______________________________________                                        Starch hydrolysis                                                                          +          R          R                                          Casein hydrolysis                                                                          +          +          +                                          Gelatin hydrolysis                                                                         +          +          +                                          Hippurate hydrolysis                                                                       ..         +          ..                                         Citrate utilisation                                                                        ..         +          ..                                         Catalase     -          -          -                                          Oxidase      -          +          +                                          Growth in 3% saline                                                                        w          w          -                                          Sugar fermentations:                                                          Galactose    w          -          -                                          Glycogen     +          w          -                                          Mannitol     -          w          +                                          Raffinose    +          +          w                                          Starch       +          -          -                                          Trehalose    +          w          +                                          Xylose       +          -          -                                          ______________________________________                                                     B.        B. coagulans                                                                             B. coagulans                                             caldolyticus                                                                            ATCC 8083  ATCC 12245                                  ______________________________________                                        Starch hydrolysis                                                                          +         -          -                                           Casein hydrolysis                                                                          +         -          w                                           Gelatin hydrolysis                                                                         +         -          -                                           Hippurate hydrolysis                                                                       ..        ..         ..                                          Citrate utilisation                                                                        ..        ..         ..                                          Catalase     -         +          +                                           Oxidase      +         -          -                                           Growth in 3% saline                                                                        -         -          -                                           Sugar fermentations:                                                          Galactose    -         +          +                                           Glycogen     +         w          -                                           Mannitol     w         w          -                                           Raffinose    -         -          -                                           Starch       +         w          w                                           Trehalose    +         +          +                                           Xylose       -         w          w                                           ______________________________________                                         Test were performed in parallel according to Sharp, R. J., Bown, K. J. an     Atkinson R. (1980) on B. stearothermophilus strain LLD15, LLDR and NCA        1503 and B. caldotenax. The other data are from that reference; R,            restricted; +, positive; w, weak positive; -, negative; .., not tested.  

LIST OF FIGURES

The text refers to FIGS. 1-6 which are as follows:

FIG. 1--Anaerobic pathways and theoretical yields in Bacillusstearothermophilus strain LLD-15

FIG. 2--Steady state values in continuous cultures at pH7, D=0.2 hr⁻¹ onsucrose 10 g/1 anaerobically or 5 g/1 aerobically (dotted line), 0.5%tryptone, 0.25% yeast extract

(a) Biomass: ( ) strain NCA 1503, ( )strain LLD-R, (◯) strain LLD-15

(b) Products with strain LLD-15

FIG. 3--Continuous cultures of strain LLD-15 on various concentrationsof wheat-straw hydrolysate (Ragg and Fields, 1986) at 70° C., D=0.2hr⁻¹,pH7.

FIG. 4--A continuous two-stage reactor system for ethanol production.

FIG. 5--Continuous fermentation with partial cell recycle, plant.

FIG. 6--Continuous fermentation with partial cell recycle,relationships.

ANAEROBIC PATHWAYS IN THE NEW STRAINS

Our original experiments were conducted mostly at 60° C. in batchculture on 2.35% w/v) sucrose/BST medium since that is the optimaltemperature for strain NCA 1503 which was the presumed ancestor of themutant strain LLD-15. In anaerobic batch cultures of strain NCA 1503 orstrain LLD-R the final product is predominantly L-lactate, whereasstrain LLD-15 gives (moles/mole sucrose): ethanol (1.8), acetate (1.8)and formate (3.2) at pH 7.9. This is consistent with metabolism via thepyruvate-formate lyase (PFL) pathway (FIG. 1) since the mutationabolishes L-lactate dehydrogenase activity. However at more acid pH theratio of ethanol to acetate increases becoming at pH 6.2: ethanol (2.9),acetate (0.2) and formate (1.3). This indicates that a novel pathwayleading to 2 ethanol+2 CO₂ from each glucose residue is also operating,and we believe that this is via pyruvate dehydrogenase (PDH) which isgenerally considered to be inoperative anaerobically. In summary theproducts in FIG. 1 are:

    ______________________________________                                                       Products (moles/mole sugar)                                    Pathway        Ethanol Acetate Formate CO.sub.2 ATP                           ______________________________________                                        Glucose:                                                                      Glycolysis + PDH                                                                             2.00    0.00   0.00  2.00 2.00                                 Glycolysis + PFL                                                                             1.00    1.00   2.00  0.00 3.00                                 Entner-Doudoroff + PDH                                                                       1.50    0.50   0.00  0.00 3.00                                 Entner-Doudoroff + PFL                                                                       1.00    1.00   1.00  1.00 2.00                                 Xylose:                                                                       Pentose cycle + PDH                                                                          1.67    0.00   0.00  1.67 1.67                                 Pentose cycle + PFL                                                                          0.83    0.83   1.67  0.00 2.50                                 Phosphoketolase + PDH                                                                        1.00    1.00   0.00  1.00 1.50                                 Phosphoketolase + PFL                                                                        0.50    1.50   1.00  0.00 2.50                                 ______________________________________                                    

The switch from the PFL- to the PDH-pathway occurs in the later stagesof anaerobic batch fermentations with strain LLD-15 and is associatedwith a slowing in growth rate and the appearance of traces of pyruvatein the medium. The effect is most marked during batch fermentations onhigh sugars, e.g. 5% (w/v) sucrose, where growth ceases long before allthe sugar is utilised but the non-growing cells continue to convertsucrose quantitatively to ethanol+CO₂. Hence in such a batchfermentation ethanol yields can reach 3.64 moles/mole sucrose (91%theoretical). Higher temperature (70° C.) also favours a switch to thePDH-pathway.

The switch from the PFL to the PDH pathway is due to accumulation ofacetate and formate in the medium, and not due to ethanol accumulation,as shown by adding these products to the medium in the early stages of abatch fermentation. Pyruvate secretion is observed whenever thePDH-pathway is significantly operative. Cells grown under suchconditions show levels of pyruvate dehydrogenase activity in cell-freeextracts even higher than those in fully aerobic cells, whereas thewild-type shows very low anaerobic PDH levels. There is no detectablepyruvate decarboxylase or formate dehydrogenase activity, which mighthave provided an alternative pathway for ethanol and CO₂ production. Theswitch in pathways may be a pre-sporulation phenomenon as biomassdecreases towards the end of batch fermentations and spores areobserved.

The probable reason for the switch in pathways is as follows. The wildtype organism is geared to rapid growth, both aerobic and anaerobic, sohas rapidly acting sugar uptake and glycolysis systems. It normallysecretes L-lactate under anaerobic conditions, but when that pathway isblocked pyruvate metabolism is shunted into the PFL-pathway. However therate of excretion of acetate and formate then becomes the rate-limitingstep for energy metabolism, particularly in the presence of externalacetate and formate or at acid pH where secretion against an anion orproton gradient reduces the efflux rate. Hence pyruvate accumulateswithin the cell and induces the pyruvate dehydrogenase activity tolevels even higher than in fully aerobic cells. The flux throughpyruvate dehydrogenase is however still inadequate to maintain the rapidgrowth rate seen at alkaline pH or at low sugar concentration in theabsence of acetate and formate, and the cells reach a stationary stagein which sugars are converted quantitatively to ethanol and CO₂ withoutgrowth.

SINGLE STATE CONTINUOUS CULTURES

Preliminary experiments to compare wild type (strain LLD-R) and strainLLD-15 were conducted with 2-3% sucrose in BST medium at 60° C.(dilution rate 0.25 hr⁻¹). As expected, wild type cells producedpredominantly L-lactate, ranging from 3.13 moles/mole sucrose consumedat pH8 to 3.50 at pH 6.35 and Y values (g.cells/g.sucrose) were around0.07. With the mutant strain at pH7 ethanol was the major product (2.3moles/mole sucrose) and the Y value was higher (0.10). However strainLLD-15 was unstable in continuous cultures at acid pH or at high sugarconcentrations, and takeover by revertants to L-lactate production(strain LLD-R) was common. This reflects the powerful selection pressurefor increased energy efficiency exerted by such continuous cultures, andillustrates a potential defect of a continuous process. However thisreversion is less frequent in continuous cultures at 70° C. on lowersugar concentrations and can be eliminated by reselection from strainLLD-R of a non-reverting mutant (procedure of Payton and Hartley3).

FIG. 2a shows the steady-state biomass in continuous cultures of strainsNCA 1503, LLD-R and LLD-15 grown anaerobically on 1% (w/v) sucrose oraerobically on 0.5% sucrose, 0.5% tryptone, 0.25% yeast extract atpH7.0, dilution rate 0.2 hr⁻¹, at various temperatures. Both the newstrains and strain NCA 1503 show efficient aerobic and anaerobicmetabolism, but strain NCA 1503 expires anaerobically above 70° C.whereas both the wild-type (LLD-R) and the mutant (LLD-15) retainsignificant anaerobic metabolism up to 75° C. This temperature is closerto the boiling point of aqueous ethanol and is therefore significant forthe processes described herein.

The products from the anaerobic continuous cultures of strain LLD-15 areshown in FIG. 2b. It is clear that ethanol productivity (mmol/A₆₀₀)rises as temperature increases and is associated with pyruvatesecretion.

Strain NCA 1503 does not grow on xylose either aerobically oranaerobically, but both LLD-R and LLD-15 do. The results with sucrosecan be compared with continuous cultures on 1% xylose at 70° C. inanalogous conditions (Table 2). Steady states can again be maintainedand alcohol yields are again higher at acid pH.

                  TABLE 2                                                         ______________________________________                                        Continuous anaerobic cultures of strain LLD-15 on                             xylose (10 g/l), tryptone (5 g/l), yeast extract                              (2.5 g/l), BST salts at 70° C., D = 0.2 hr.sup.-1                              Residual Products:                                                    Cells   Xylose   (moles/mole xylose, g/g cells/hr)                            pH  (g/l)   (g.l)    Ethanol  Acetate Formate                                 ______________________________________                                        6.5 0.35    0.9      1.70 (0.13)                                                                            1.79 (0.17)                                                                           3.48 (0.25)                             7.0 0.98    0.9      1.43 (0.04)                                                                            2.14 (0.07)                                                                           3.84 (0.10)                             8.0 0.67    0.9      0.82 (0.03)                                                                            1.07 (0.05)                                                                           2.20 (0.08)                             ______________________________________                                    

The steady state biomass at pH7 is less than that with the concentrationof sucrose so xylose metabolism is less energy efficient. Sucrose isbelieved to be metabolised to two hexose phosphates via a relevantkinase+phosphorylase, requiring one ATP. In contrast two ATP's arebelieved necessary to product two pentose phosphate molecules. Hencesucrose is intrinsically a better energy substrate.

The products of the continuous xylose fermentations show that anappreciable proportion of the tryptone-yeast extract is metabolised forenergy production. Nevertheless the product ratios at pH8 indicate thatmetabolism proceeds via the pentose phosphate pathway, glycolysis andthe PFL-pathway. The ethanol yields are lower than on sucrose,suggesting little flux through the PDH-pathway. However fermentations onhigher sugar concentrations at higher temperatures and lower pH areexpected to increase the flux through the latter pathway, as forsucrose, and mutations in acetate kinase or phosphotransacetylase toproduce a non-growing strain that converts xylose quantitatively toethanol and CO₂.

Hence the new strain and derivatives thereof are an organism of choicefor ethanol production from hydrolysates of lignocellulosic wastes.Continuous cultures have been performed on crude hydrolysates of wheatstraw produced by the ICI hydrolysis process described by Ragg andFields⁴. This is effectively a waste stream, rich in xylose and lignin,produced by a short dilute acid hydrolysis step designed to removehemicelluloses and thereby facilitate subsequent delignification. Thecrude material is adjusted to pH7.0 and tested in continuous culturewith the new strain at 70° C., D=0.2 hr ⁻¹ at various dilutions as shownin FIG. 3. The waste-stream provides all necessary nutrients forcontinuous culture of the organism and all the sugars are utilised tosome extent. Ethanol yields increase at lower pH and the product ratiosare consistent with metabolism via the pentose phosphate pathway,glycolysis and PFL plus PDH (see FIG. 1).

A TWO-STAGE AEROBIC/ANAEROBIC FERMENTATION

The property of quantitative conversion of sugars to ethanol withoutgrowth is a significant potential advantage of the new strain. It can bemaximised by further manipulation of physiological constraints, asillustrated above, or by selection of further mutations. FIG. 1 showsthat cells lacking acetate kinase or acetyl CoA-phosphotransacetylasecannot produce acetate. Since acetate secretion is essential to maintainanaerobic flux through PFL, only the PDH-pathway remains open forpyruvate metabolism resulting in ethanol+CO₂. Such cells may not growanaerobically but can be produced aerobically and used anaerobically toconvert sugars to ethanol catalytically.

Moreover we have seen that mutations that increase intracellularpyruvate dehydrogenase activity will increase ethanol productivity,since PDH appears to limit energy flux. Such mutations will be selected,either spontaneously or after mutagenesis, by growth in continuousculture or on plates under conditions in which intracellular acetate andformate accumulation occur, i.e. on sugars at low pH+added acetate andformate. Alternatively additional copies of the PDH-genes can beintroduced by genetic engineering protocols.

Since maximum ethanol productivity is associated with cessation ofgrowth, conventional anaerobic batch cultures are unsuitable for ethanolproduction by such strains. Batch production may be achieved byintroducing a large inoculum of cells grown aerobically into theanaerobic reactor, or by conducting batch fermentations under conditionsof partial anaerobiosis, where the total biomass will depend on thelevel of oxygen supplied.

Moreover an indefinite continuous process catalysed by non-growing cellsis clearly impossible; a minimum uptake of sugar (maintenancecoefficient, m₃) is needed to maintain cell viability. We have seen thatthis can be achieved in a single-stage anaerobic reactor with partialcell recycle such as that illustrated in FIG. 5 without recycle orbleed; the system operates as a conventional single-stage continuousculture through the level controller system. When this is blocked andrecycle begins, biomass levels rise to a maximum dictated by themaintenance coefficient. Thereafter all substrate is converted toproducts. This is clearly advantageous for production purposes, but willin practice lead to steadily decreasing (m) reactor productivity.However if a small bleed is taken from the reactor (F_(x)), steady-stategrowth occurs at a rate μ=F_(x) /V (where V=reactor volume). This can beminimised to balance declining reactor productivity. In the figure,sugars and nutrients are pumped in at a rate F_(i). A constant bleedF_(x),(F_(x) <<F_(i)) determines the cell growth rate μ/=F_(x) /V,(V=fermentor volume). The remaining broth is recirculated through ahollow fiber ultrafiltration membrane, operated at its maximum capacity.The filtrate output F_(f) is controlled by a level controller system;the excess filtrate is returned to the fermentor.

FIG. 6 shows the results of a model system with strain LLD-15 on 1%sucrose/BST AM at 70° C., pH 7. The figure shows the relation betweenvolumetric ethanol productivity, cell concentration and total dilutionrate, D=F_(l) /V. S_(o) =1%, T 70° C., 400 rpm, pH 7.0 Bacillusstearothermophilus LLD-15. Cell growth rate μ=F_(x) /V=0.1 h⁻¹. Thegrowth rate was kept constant at 0.1 h⁻¹, by fixing the bleed rateF_(x). The overall dilution rate D was increased by increasing thesugars and nutrients feed rate F_(i). Sucrose consumption (not shown)was always above 97% indicating a high stability of the system. Thevolumetric ethanol productivities were significantly higher thanconventional single stage continuous fermentations (i.e. 0.6 gethanol/1-h). This was primarily due to the proportional increase incell density achieved at high dilution rates.

Such reactor systems are feasible for ethanol production by thesestrains, but we have seen that the special properties of a facultativeanaerobe can be maximised for ethanol production in the novel reactorconfiguration illustrated in FIG. 4. In summary, sugars are pumped intothe anaerobic reactor A at rate V₂. Vapour phase ethanol is separatedfrom CO₂ by water absorption. A portion of spent cells is removed bycentrifugation (C) and ethanol distilled from the effluent stream. Theresidual sugars and ethanol are supplemented with nutrients (N) at rateV_(n) and used to create catalytic biomass aerobically (B). Theresulting cells are returned to reactor A after centrifugation. In moredetail sugars such as cane-juice, molasses, straw-hydrolysate, etc., arefed at rate V_(S) into an anaerobic reactor supplied by cells at rateV_(R). For the purpose of illustration the reactor A is a simple stirredtank (volume V_(A)) in which temperature and pH are controlled tomaximise ethanol yield and productivity. Ethanol in the vapour phase isseparated from CO₂ by absorption with water before continuousdistillation. However one of the major advantages of a thermophilicfermentation is that as the boiling point of aqueous ethanol isapproached it can be removed continuously and economically from theaqueous phase to remove ethanol inhibition of growth and/or productivity(in the case of strain LLD-15, growth ceases above 4% (w/v) ethanol at60° C.). This allows the use of higher concentrations of sugars asfeedstock, such as molasses. Hence the anaerobic reactor may withadvantage be one that maximises rate of ethanol removal into vapourphase such as vacuum fermentation, sparging with recycled CO₂ orcontinuous recycling through a vacuum flash evaporator.

Most of the cells in the effluent from A are concentrated by continuouscentrifugation and recycled. Then ethanol is removed from thesupernatant by continuous distillation. The stream entering the aerobicreactor B will contain spent cells (or spores), residual ethanol,unutilised sugars and by-products such as acetate and formate. Most ofthese can serve as aerobic substrates for strain LLD-15. Hence thestream is supplemented with necessary nutrients to allow maximumconversion of these waste carbon sources to biomass.

That biomass is concentrated by centrifugation and returned to theanaerobic reactor (volume V_(B)). There is a problem in that a lag phasemay be observed before aerobic cells become adapted to anaerobicmetabolism. Hence it may be advantageous to operate reactor B underoxygen limitation or to interpose an intermediate `anaerobic adaptation`reactor fed with low sugars at optimum growth pH before the cells returnto the catalytic stage.

The process variables in such a reactor configuration are complex, butthe system has a redeeming feature. Optimal ethanol yield for any givenfeed composition and rate (V_(S)) occurs when anaerobic CO₂ (=ethanol)production is maximal and aerobic CO₂ (assuming complete sugarsoxidation) is minimal. Optimal productivity is given by maximizingV_(S). Hence by using CO₂ sensors to control the pump rates, pH andtemperature in each vessel, the system lends itself toself-optimisation. This is a considerable advantage in minimizing pilotplant development work with any particular substrate and an even greateradvantage at plant scale in dealing with feedstocks of variablecomposition.

SUMMARY

In its preferred form, now summarised but without derogation from theclaims, the present invention uses mutants of an extremely thermophilicfacultative anaerobe such as the novel Bacillus stearothermophilusstrain LLD-R (NCIB 12403) capable of rapid aerobic and anaerobic growthand/or metabolism above 70° C. with a wide range of sugars includingpentoses and cellobiose arising from hydrolysis of lignocellulose. Themutants, such as strain Bacillus thermophilus LLD-15 (NCIB 12428), aredesirably selected so as to switch anaerobic pathways predominantlytowards ethanol production. These are the deposited strains referred toherein.

Strain LLD-R grows rapidly on a wide range of sugars up to 75° C. butthe major anaerobic product is L-lactate. The mutant strain LLD-15 growsequally rapidly via two major energy pathways : the pyruvate - formatelyase (PFL) pathway yielding 1 mole ethanol, 1 acetate and 2formate/mole glucose residue, and a previously unrecognised pyruvatedehydrogenase (PDH) pathway yielding 2 ethanol+2 CO₂ /mole glucose.

The metabolic flux in strain LLD-15 can be directed through thePDH-pathway by manipulation of physiological conditions, in particular abuild-up of pyruvate caused by growth at acid pH or by the presence ofacetate and formate in the medium. Higher temperatures also favour thePDH-pathway. The cells may not grow under such conditions but continueto convert sugars to ethanol. Alternatively the PDH-flux can beincreased by further mutations; for example mutations which suppressacetate production. Other desirable mutations are those which increasetotal anaerobic pyruvate dehydrogenase activity, since this israte-limiting for ethanol productivity.

Such strains are optimal for a two-stage fermentation in which catalyticbiomass is first grown in an aerobic seed stage and subsequently usedanaerobically in an ethanol production stage without growth. This can beachieved in a single-stage batch or fed-batch reactor, with continuingethanol removal in the vapour phase to allow use of concentrated sugarfeedstocks. Conventional feedstocks such as glucose, sucrose or maltosemay be used, but also sugars arising from hydrolysis of lignocellulosicwastes including pentoses and cellobiose.

The strains are not very suitable for conventional single-stagecontinuous cultures but may be used to advantage in a single stagesystem with partial cell recycle or in a two-stage continuous system inwhich sugars are fed to an anaerobic catalytic reactor with continuingethanol removal in the vapour. The remaining ethanol is stripped fromthe aqueous effluent from this reactor and the residual carbon sourcesare utilised to create new catalytic biomass in an aerobic biomassstage. Thereby effectively all of the potential substrates in thefeedstocks are utilised either anaerobically or aerobically. Moreoverthis system lends itself to automated self-optimisation for ethanolproduction by maximising anaerobically-produced CO₂ (equivalent tobiomass production). This can be a particular advantage when using mixedand variable feedstocks.

REFERENCES

1. Hartley, B. S. et. al. (1983) In `Biotech 83` Online PublicationsLtd., Northwood, U. K., p. 895.

2. Hartley, B. S. and Shama, G. (1987) In `Utilisation of CellulosicWastes` (eds. Hartley, B. S., Broda, P.M.A., and Senior, P.), The RoyalSociety, London.

3. Payton, M. A. and Hartley, B. S. (1985) FEMS Microbiol. Lett. 26,333.

4. Ragg, P. L. and Fields, P. R. In `Utilisation of LignocellulosicWastes` (Eds. Hartley, B. S., Broda P.M.A. and Senior, P.), The RoyalSociety, London.

I claim:
 1. A two-stage closed system process for the production ofethanol comprising(i) carrying out ethanol-producing anaerobicfermentation of sugars in an anaerobic fermentation medium at atemperature of at least about 70° C. in the presence of a thermophilic,facultatively anaerobic bacterium capable of fermenting sugars bothaerobically and anaerobically and producing ethanol in anaerobicfermentation at temperatures 70° C. or above; (ii) continuously removingethanol during anaerobic fermentation (i); (iii) continuouslywithdrawing a portion of the fermentation medium from anaerobicfermentation (i); (iv) separating bacteria from the withdrawnfermentation medium and recycling the separated bacteria to anaerobicfermentation (i); (v) removing ethanol from the withdrawn portion of thefermentation medium; (vi) adding the ethanol free fermentation medium toan aerobic reactor and culturing the bacteria, returning a portion ofthe resulting bacteria and the medium to the anaerobic fermentationmedium of anaerobic fermentation (i) so as to maintain catalyticbiomass.
 2. The process according to claim 1, wherein said strainproduces maximum yields of ethanol while minimizing anaerobic growth ofsaid strain.
 3. The process according to claim 1, wherein anaerobic andaerobic CO₂ production is monitored and the anaerobic CO₂ to aerobic CO₂ratio is maximized for each of the process conditions employed.
 4. Theprocess according to claim 2, wherein the strain lacks NAD-linkedlactate dehydrogenase activity.
 5. The process according to claim 1,wherein the strain produces ethanol in said anaerobic fermentation bypyruvate dehydrogenase pathway activity.
 6. The process according toclaim 2, wherein the strain during said anaerobic fermentationsuppresses pyruvateformate lyase pathway flux.
 7. The process accordingto claim 1, wherein said sugars comprise pentoses or cellobiose.
 8. Theprocess according to claim 1, wherein the thermophilic, faculativelyanaerobic bacterium is a strain of Bacillus stearothermophilus.
 9. Theprocess according to claim 8 wherein the strain is selected from thegroup consisting of B. stearothermophilus LLD-R (NCIB 12403) and B.stearothermophilus LLD-15 (NCIB 12428) and ethanol producing variantsand derivatives thereof.